Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy

Abstract Background Cells and tissues, such as macrophages, express inducible nitric oxide synthase (INOS) after stimulation by certain factors. INOS helps mediate the macrophage inflammatory reaction, but few studies have explored how INOS affects macrophage function in nonalcoholic fatty liver disease (NAFLD). Objective This study investigated the role of INOS‐mediated macrophage activity in NAFLD. Methods A high‐fat diet was used to establish an NAFLD mouse model. After 12 weeks, blood was collected for immune cell and lipid analyses, and liver tissues were collected for pathological analyses with hematoxylin and eosin and Oil Red O staining. Peritoneal macrophages were extracted in situ, cultured in Dulbecco's modified Eagle's medium, and stimulated with palmitic acid to mimic in vivo conditions for further assays. Real‐time polymerase chain reaction, western blot analysis, and immunofluorescence were used to verify the expression of target genes or proteins. Results In the NAFLD model, INOS expression in macrophages increased, and INOS knockdown significantly decreased the number of macrophages. Pathological examinations confirmed that INOS knockdown slowed NAFLD progression and macrophage infiltration during inflammation. INOS knockdown also enhanced phagocytosis and lipid transport by macrophages, and increased the expression of autophagy‐related molecules in macrophages, which improved the autophagy level, promoted apoptotic cell degradation, and maintained intracellular environment homeostasis. Conclusions These results indicate a correlation between INOS expression and macrophage function in NAFLD.


| INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is a pathological metabolic disease caused by several factors (excluding alcohol or liver injury), such as metabolism, gene expression, environmental pollution, and intestinal microorganisms.The incidence of NAFLD has increased in many developing countries, 1 occurring in approximately 24% of adults worldwide.However, this value is often underestimated owing to detection method limitations. 2,3NAFLD includes simple fatty liver and nonalcoholic steatohepatitis (NASH), which can progress to liver fibrosis and cirrhosis, increasing the risk of hepatocellular carcinoma.NAFLD is closely associated with various metabolic diseases, including obesity, insulin resistance, type 2 diabetes, and dyslipidemia, 4,5 and except for lifestyle interventions and weight loss, clear and effective treatments do not exist. 6,7herefore, exploring the pathogenesis of NAFLD may provide new insights into clinical therapies.
The pathogenesis of NAFLD remains widely debated and mainly includes the "second strike" theory, fructose induction, adipose tissue dysfunction, intestinal flora disorders, abnormal bile acid metabolism, and the accumulation or lack of metal elements. 8The hepatic immune microenvironment, including Kupffer cells, is key in NAFLD progression.Kupffer cells (immune cells) settle in the liver, and recruited macrophage polarization plays a key role in the pathogenesis of liver inflammation, fibrosis, and NAFLD.Hepatocyte injury may induce macrophage polarization into the classic activated macrophage M1 type, secrete proinflammatory cytokines and other substances, and recruit more monocyte macrophages to aggravate liver injury. 9veral factors and pathways promote macrophage activation in NAFLD.First, plasma endotoxin levels increase as intestinal microbiota changes, intestinal bacterial overgrowth, and increased intestinal permeability occur in patients with NAFLD; these events are related to the gut-liver axis. 102][13] Second, hepatocyte damage triggers macrophage activation, and apoptotic bodies activate Kupffer cells. 14Specific oxidized epitopes on apoptotic cells or cell fragments act as risk molecular patterns, which also activate macrophages through pattern recognition receptors. 157][18] Third, free fatty acids and leptin secreted by adipose tissue activate Kupffer cells through TLRs and leptin receptors. 19,20oreover, increasing evidence suggests that excess cholesterol (CHO) is a potential pathogenic cofactor of NAFLD.CHO and its metabolites activate Kupffer cells through cluster of differentiation (CD) 36 and scavenger receptor A. 21 Finally, Kupffer cells secrete proinflammatory cytokines and chemokines, which recruit circulating neutrophils and monocytes to the liver.Monocytes differentiate into proinflammatory macrophages, which amplify liver inflammation and stimulates stellate cell transformation into activated myofibroblasts, leading to fibrosis.In conclusion, functional changes of different macrophages play important regulatory roles in NAFLD development.Therefore, this study focused on changes in immune cells and macrophage function in an NAFLD disease model to further elucidate the relevant mechanisms.
Nitric oxide synthase (NOS), generally classified as constitutive NOS (cNOS) and inducible NOS (INOS), is distributed among various cells.cNOS is sub-classified as neural NOS and endothelial NOS (eNOS).Generally, INOS is not expressed in cells; it is primarily found in macrophages stimulated by LPS, cytokines, and other factors.Once expressed, INOS remains active and independent of the intracellular Ca 2+ concentration.eNOS and INOS exist in the liver; specifically, eNOS exists in intrahepatic blood vessels, sinusoidal endothelial cells, and hepatocytes, and INOS exists in hepatocytes and hepatic macrophages.INOS mainly produces nitric oxide (NO) at the transcriptional level, which is a free radical gas with extremely active chemical properties with a dual oxidation and antioxidation role in the oxidative stress response of tissues and organs.NO and INOS levels are significantly associated with the occurrence and development of NAFLD.Both promote lipid peroxidation in the liver, inhibit hepatocyte protein synthesis, cause glucose metabolism disorders, promote mitochondrial damage, and accelerate hepatocyte apoptosis. 22onsidering the importance of macrophages and the uncertainties surrounding the pathogenic mechanisms of NAFLD, this study investigated how INOS in macrophages affects NALFD, which may have clinical significance for treating this disease.

| Animal experiments
All mice were housed at the Center for Experimental Animals of the Medical Research Centre of the Affiliated Hospital of Jining Medical University under pathogenfree conditions throughout the experiment.C57BL/6J male mice (18-20 g, 6-7 weeks old) were purchased from Weitonglihua Co. and housed in plastic cages lined with sawdust.The animals were kept under a 12-h light-dark cycle, and food and water were provided ad libitum.
The mice were randomly assigned to control or methionine-and choline-deficient (MCD) diet groups (n = 6 per group).The control mice were fed a normal diet, and the MCD diet group was fed an MCD diet.MCD food was purchased from Medicine Professionals for Laboratory Animal Diets.The animals were fed their respective diets for 2 weeks (14 days), then all mice were euthanized under anesthesia (intraperitoneal pentobarbital injection), and blood and liver samples were collected.
In addition, we obtained mice from the Immunology Department of Basic Medical Science of Jining Medical University to create male congenic wild-type (INOS +/+ ) and INOS knockout (INOS −/− ) mice.Then, the INOS +/+ and INOS −/− mice were fed a high-fat diet (HFD) for 12 weeks and then euthanized with carbon dioxide.Blood samples were collected for lipid and immune cell analyses.The livers were also dissected, frozen, and fixed in 4% paraformaldehyde overnight to prepare sections and then stained with Oil Red O for observation under an optical microscope or subjected to immunofluorescence staining.
All laboratory animals were housed in regulated rooms, and all operations and experiments were conducted following the National Institutes of Health Guide for Care and Use of Laboratory Animals.

| Histopathology
Liver tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into thin slices for hematoxylin and eosin (HE) staining.In addition, 5 µm of frozen liver sections were fixed and stained with Oil Red O (Sigma-Aldrich).

| Flow cytometry
Cells were isolated from the blood and liver samples and then stained for CD3, CD4, CD8, F4/80, and CD11c (eBioscience) and analyzed by flow cytometry using CytoFLEX S (Beckman Coulter).
The cells were then washed and treated with an antifluorescence quencher, as determined using laser scanning confocal microscopy (LSM780; Zeiss).

| Quantitative real-time PCR (qRT-PCR)
Peritoneal macrophages from INOS +/+ or INOS −/− mice were isolated and cultured, and the cells were stimulated with PA.Total RNA was extracted using TRIzol reagent (Invitrogen/Thermo Fisher Scientific) and used for complementary DNA synthesis with the PrimeScript TM RT Reagent Kit with DNA Eraser (TaKaRa) following the manufacturer's instructions.qRT-PCR for the genes of interest was performed using a LightCycler CFX96 (Bio-Rad) under the conditions: preincubation at 95°C for 10 s, followed by 40 cycles of 95°C for 2 s, 55°C for 5 s, 72°C for 35 s, then 72°C for 5 min.Table 1 lists the specific primers.
Each experiment was performed in triplicate.Transcript levels of the target genes were calculated by the ∆∆ 2 C -T method.All primers were synthesized by the Beijing Genomics Institution.

| Statistical analyses
Statistical differences between any two groups were evaluated using the independent sample double-test t test, and the values were expressed as means ± standard deviations.Each experiment was performed at least three times.p values of <.05 were considered statistically significant.All statistical analyses were performed using GraphPad Prism 5.0 software (Graph-Pad Software).

| NAFLD microenvironments enhance INOS expression in macrophages
The livers of the MCD diet model group were yellow and felt greasy compared to those of the control (normal diet) group.Furthermore, HE and Oil Red O staining indicated widespread steatosis.Increased serum ALT concentrations were also observed, indicating that the model was successfully created (Figure 1A-C).WB and immunofluorescence analyses also confirmed that INOS expression in the liver tissues was significantly higher in the NAFLD model group than in the control group (Figure 1D,E).
To explore the changes in the liver's immune microenvironment during NAFLD progression, we evaluated the number of immune cells in the liver and peripheral blood, including dendritic cells, T cells, and macrophages.The flow cytometry results indicated that the number of immune cells significantly differed between the NAFLD and control groups, and macrophages differed the most (Figure 2A-E).We also confirmed enhanced INOS expression in the macrophages in these models.PA stimulation of macrophages in vitro (to mimic the NAFLD microenvironment) resulted in a significant and time-dependent increase in INOS expression in the PA-stimulated macrophages, confirmed by WB and qRT-PCR (Figure 2F-H).These data strongly indicate that the NAFLD microenvironment enhanced INOS expression in macrophages.

| INOS −/− alleviates steatosis with a HFD diet
To assess the effects of increased INOS expression in the NAFLD models, we developed an HFD diet-induced NAFLD model in INOS +/+ and INOS −/− mice that had been genotyped by PCR (Supporting Information S1: Figure 1 presents a representative genotype).After 4 months on an HFD, the INOS −/− mice exhibited markedly minor lipid accumulation (Figure 3A), palliative characteristic hepatic ballooning vacuolization in HE-stained sections, less lipid deposition in Oil Red O-stained sections (Figure 3B), and lower ALT, AST, CHO, TG, and LDL-C concentrations (Figure 3C-G) compared to the controls, demonstrating that INOS knockout alleviated HFD-induced liver injury.Next, we performed a flow cytometry assay to further explore the relationship between improved NAFLD symptoms and macrophages, finding significant differences in the number of macrophages between the INOS −/− and INOS +/+ mice; specifically, the INOS −/− group had significantly fewer macrophages than the INOS +/+ group (Figure 4A-C).Previous studies have reported increased numbers of macrophages in the liver and peripheral blood under pathological conditions compared to the control groups, but INOS knockout reversed these effects and alleviated injury,

| INOS −/− mice have fewer M1 macrophages
In vitro, INOS knockout mitigated the increased levels of inflammatory factors after PA stimulation.Thus, we used an NAFLD disease model to further explore the effects of INOS on macrophages, finding that the number of M1 macrophages decreased significantly after INOS knockout in the liver, but the number of M2 macrophages did not differ between the knockout and control mice (Supporting Information S1: Figure 2).
Previous studies reported that M1 macrophages promote inflammation in NAFLD, and INOS regulates M1 macrophage growth and development.Thus, we detected the types of macrophages in the spleen and peripheral blood, finding that the number of M1 macrophages decreased significantly after INOS knockout (Supporting Information S1: Figures 3 and 4).Therefore, decreased levels of inflammatory factors after INOS knockout in NAFLD might be due to decreased levels of M1 macrophages resulting from INOS knockdown.

| Macrophages from INOS −/− mice have enhanced phagocytic ability
Under pathological conditions, monocytes recruited from the blood enter the liver and phagocytose to slow disease progression.In this study, we observed significantly more Oil Red O-positive cells in INOS −/− macrophages than in INOS +/+ macrophages, especially after PA stimulation (Figure 6A,B).To explain this phenomenon, we measured the expression levels of phagocytosis-related receptors, finding markedly upregulated CD36 and PPARα expression in INOS −/− macrophages (Figure 6C,D).Receptors expressed on monocytes/macrophages are associated with lipoprotein uptake, and PPARα plays a crucial role in lipid metabolism (e.g., lipogenesis, lipolysis, lipid transport, and oxidation), amino acid metabolism, gluconeogenesis, and ketogenesis; thus, it has considerable potential as a treatment target for obesity and diabetes.Furthermore, ABCA1 (a receptor related to reverse CHO transport) and CPT1A expressions were significantly higher in INOS −/− macrophages than in INOS +/+ macrophages (Figure 6F,G).Increased CPT1A expression improves biological particle uptake, suggesting that INOS participates in NAFLD by regulating lipid homeostasis.

| INOS expression increases in hepatic macrophages and correlates with autophagy
To explore the relationship between INOS expression in the liver and macrophage autophagy in NAFLD, we used an MCD diet to generate an NAFLD mouse model and then collected liver tissues for immunofluorescence staining.INOS expression was significantly higher in the NAFLD model group than in the control group (Figure 7A).Furthermore, LC3B expression was detected in the liver, suggesting a correlation between INOS and LC3B (Figure 7A) and that INOS might be involved in autophagy progression.In addition, the autophagy levels of macrophages changed significantly under pathological conditions (Figure 7B).Therefore, INOS may participate in and regulate the autophagy level of macrophages, contributing to disease progression.Dietary structures have changed with rapid lifestyle and social economy changes, which has increased the incidence of NAFLD annually.Consequently, NAFLD has become the main cause of chronic liver disease worldwide and seriously threatens public health.Prospective studies suggest that NAFLD will become the most important cause of end-stage liver disease and liver transplantation in the next decade, severely burdening the national social economy. 1 The specific mechanisms of NAFLD development have not been fully elucidated, but our study found significantly increased INOS levels in an NAFLD model.INOS is closely related to the immune response, oxidative damage, and the inflammatory response. 23Notably, INOS is mainly expressed in macrophages and is inextricably linked to NAFLD progression, particularly those in the liver tissue, including aboriginal Kupffer cells and monocytes from the blood circulation. 22Our study found that the number of macrophages in the peripheral blood and liver of mice with NAFLD increased significantly.Furthermore, in vitro macrophage stimulation by PA increased INOS expression.These results suggest that INOS helps regulate NAFLD development and progression.
We constructed an NAFLD model in mice with and without INOS, finding that INOS knockout slowed NAFLD progression but decreased the number of macrophages in the peripheral blood and liver.Studies have reported that INOS regulates macrophage production 24 ; therefore, it is plausible that the loss of INOS reduces macrophage production.We also confirmed that macrophages without INOS had significantly lower levels of related inflammatory factors stimulated by PA.The phagocytic function of the macrophages was also explored, finding that INOS knockdown regulated CD36, PPARα, and ABCA1 expression in macrophages.CD36 and PPARα in macrophages participate in lipid uptake.Moreover, ABCA1 dysfunction promotes large CHO deposits in macrophages, forming foam cells, which affects macrophage-related functions; we found upregulated ABCA1 expression after INOS knockout.Moreover, increased CPT1A expression restores macrophage phagocytosis, and CPT1A knockdown in macrophages harms macrophage phagocytosis.Our study found increased CPT1A levels after INOS knockout, confirming our hypothesis that INOS participates in and regulates the macrophage phagocytosis of lipids.In this process, INOS promotes macrophage lipid phagocytosis and CHO transport to promote the treatment of lipids by macrophages and slow the fatty liver process.
Autophagy is a "self-eating" phenomenon common in most eukaryotic cells, which is the phagocytic degradation and recycling mechanism of lysosomes to its own structure.Autophagy eliminates cells' metabolic waste via autophagy bodies and lysosome binding.Autophagy is also a protective mechanism as it helps maintain homeostasis and selfregulation in response to stress.Under certain stress conditions, the autophagy of cells is enhanced, which helps prevent or slow the progression of some diseases.Autophagy is strongly associated with the pathogenesis of NAFLD, including cellular glucose and lipid metabolism regulation, insulin sensitivity, mediating hepatocyte resistance to damaging stimuli, and preventing overactivation of the innate immune response, which are crucial for the occurrence and development of NAFLD. 26Autophagy is defective in NAFLD liver tissues, and in patients with NASH and HFD diet-induced NAFLD mouse models, the autophagy level in liver macrophages decreases, promoting the phagocytosis of apoptotic hepatocytes by macrophages.Therefore, targeting macrophage autophagy might be a new strategy for treating NAFLD metabolism and inflammation-related diseases.Studies have demonstrated that autophagy regulates INOS expression and inflammation levels.However, none have demonstrated whether INOS regulates autophagy.Our study found that the macrophage autophagy level increased in the absence of INOS, promoting the metabolism of macrophages to dead cells and lipids and slowing NAFLD progression (Figure 9).This study has certain limitations.Liver macrophages are known as Kupffer cells.Thus, to better understand the function of macrophages in the liver, Kupffer cells should be selected for further studies.In addition, the correlation between INOS and autophagy was not verified in the animal models and requires further exploration.

| CONCLUSION
This study confirms that INOS is involved in NAFLD progression by influencing macrophage-mediated changes in lipid metabolism and autophagy, providing a new research basis for treating NAFLD.

F
I G U R E 2 The NAFLD microenvironment enhances INOS expression in macrophages.(A) The percentage of CD3 + CD4 + and CD3 + CD8 + T cells from the peripheral blood of control and NAFLD mice (flow cytometry).(B-E) The numbers of F4/80 + macrophages and CD11c + dendritic cells from the peripheral blood and liver of control and NALFD mice (flow cytometry).(F-H) INOS mRNA and protein levels in PA-stimulated primary peritoneal macrophages.*p < .05,**p < .01,and ***p < .001versus the control group.CD, cluster of differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; INOS, inducible nitric oxide synthase; mRNA, messenger RNA; NAFLD, nonalcoholic fatty liver disease; PA, palmitic acid.

3. 7 |
INOS −/− macrophages have enhanced autophagy flux Primary peritoneal macrophages were isolated from INOS −/− and INOS +/+ mice, then stimulated with PA to further explore the effects of INOS on macrophage autophagy.Immunofluorescence staining demonstrated that LC3B expression was significantly higher in primary peritoneal macrophages derived from INOS −/− mice than from INOS +/+ mice (Figure 8A).We also detected autophagy flow through the mCherry-EGFP-LC3 adenovirus to confirm the regulatory effects of INOS on autophagy flux, finding enhanced autophagy after INOS knockout (Figure 8B).Furthermore, based on P62 and LC3A/B protein expression, INOS expression increased and LC3A/B expression decreased as the PA stimulation time increased (Figure 8C).However, after INOS knockout, the autophagy level significantly increased with PA stimulation (Figure 8C), suggesting that INOS is involved in macrophage autophagy under PA stimulation.F I G U R E 7 INOS expression increases in hepatic macrophages from mice with MCD-induced NAFLD and correlates with autophagy.Immunofluorescence staining of liver tissue from wild-type and NAFLD mice for co-expressing (A) INOS and LC3B and (B) Mac-3 and LC3B.Scale bar: 20 μm.DAPI, 4′,6-diamidino-2-phenylindole; INOS, inducible nitric oxide synthase; LC3B, light chain 3B; MCD, methionine-and choline-deficient; NAFLD, nonalcoholic fatty liver disease.To elucidate how INOS regulates macrophage autophagy, we measured the levels of key mTOR signaling pathway molecules after PA treatment of primary peritoneal macrophages from INOS −/− and INOS +/+ mice.P-mTOR decreased significantly after INOS knockdown (Figure8D), demonstrating that INOS affects autophagy in macrophages through the mTOR signaling pathway.

F
I G U R E 9 Macrophage autophagy level increased and decreased levels of inflammatory cytokines in the absence of INOS, promoting the metabolism of macrophages to dead cells and lipids and slowing NAFLD progression.IL-1β, interleukin-1β; IL-6, interleukin-6; INOS, inducible nitric oxide synthase; Mac, macrophage; TNF-α, tumor necrosis factor-α; WT, wild-type.